Hanging Drop Culture Promote Osteogenic Differentiation Of Human Adipose-derived Stem Cells

ObjectiveThis study was designed to investigate the effect of hanging drop cultureon osteogenesis efficiency of human adipose-derived stem cells (hADSCs).MethodshADSCs were isolated by using enzymatic digestion and cultured. Theprimary culture and passage culture of human adipose derived stem cells wereobserved under the phase-contrast microscope. To confirm theimmunophenotype of hADSCs, surface markers of CD44, CD90, CD105,CD34and CD45were detected by flow cytometry. Determination of hADSCsmutiple differentiate potential into adipogenic and osteogenic cells in vitro wasestablished by exposion to different inducers. Then, hADSCs were cultured inthe hanging drops. The forming process of hADSCs3D spheroid aggregates inhanging drops were observed under phase-contrast microscope. H&E stainingof sections revealed the histology structure of spheroid aggregates. With24h、72h or96h culture of hADSCs spheroid aggregates of10,000,25,000or10,000, viability of spheroid aggregates was measured by live/dead, andcompared the sizes of hADSCs spheroid aggregates. Analysis the effect ofhanging drops culture and plate culture on proliferation useing MTT andCFU-F assays of hADSCs. By using flow cytometry detected the variation ofsurface markers expression on hADSCs(2D or3D) and identified the hADSCs capability of differentiation potential into adipogenic and osteogenic cells. Aftercoculture for7,14or21days, the cells were digested for isolation of total RNA.Real time fluorescence quantitative polymerase chain reaction (PCR) wasused to detect the relative mRNA levels of collagen type I, osteopontin, andosteocalcin, all of which are the marker genes for osteoblasts. The expressionof collagen type I, osteocalcin and osteopontin was detected by PCR at7,14and21days.ResultshADSCs, isolated and cultured by enzymatic digestion, sharedimmunophenotype of both multipotential stem cell and embryonic stem cellmaker, as they were positive for CD44, CD90, CD105, and negative for CD34and CD45expressions by flow cytometry analysis. hADSCs had multipledifferentiate potential into adipogenic and osteogenic cells in vitro exposed todifferent inducers; hADSCs cultured in the hanging drops can formthree-dimensional spheroid aggregates with uniform size after96h of culture.With the increase of cultured time, the diameter of spheroid aggregatesreduced, and arregates became closer and more compact. Cell viability testrevealed that viability were highest with hADSCs of25,000and72h of hangingdrop culture. Cell viability of spheroid aggregates reduced over time;Compared with plated culture, hADSCs cultured in the hanging drops hadmuch higher capability of cell proliferation and clone. The result of flowcytometry analysis revealed that there is no difference of surface marker inhADSCs cultured in the hanging drops and plate. hADSCs cultured in thehanging drops had multiple differentiate potential into adipogenic andosteogenic cells, and it indicated that hanging drops culture had no effect ofbasic stem cell characteristic of hADSCs; PCR showed that the expressionlevels of Collagen type I,Osteocalcin and Osteopontin in hanging drop cultureswere much higher than that of control. Hanging drop culture can promoteosteogenic differentiation of hADSCs.. ConclusionsHanging drop culture is benefit for the efficiency of proliferation andinduced osteogenic differentiation of human adipose-derived stem cells.